Potassium balance is ultimately dependent on potassium channels in the renal cortical collecting duct. These channels are exquisitely regulated, ensuring that precisely enough potassium is secreted into the tubule lumen to match dietary intake. The asymmetric expression of two different potassium channels increases the fidelity of the secretory process. K_ATP channels, exclusively expressed on the apical membrane, are designed to allow avid potassium efflux into the tubule lumen. In contrast, the basolateral membrane channels, Kir2.3, are strongly inwardly rectifying, limiting futile recycling of potassium into the interstitium. Here we show that these channels are regulated, targeted and retained on polarized membrane domains as parts of multimeric complexes that are co-ordinated by PDZ-domain-containing proteins.

We found that the basolateral-sorting determinant in Kir2.3 comprises a unique arrangement of trafficking motifs, containing tandem, conceivably overlapping, biosynthetic targeting and PDZ-based retention signals. Deletion of the entire sorting cassette produced channels that are mistargeted and expressed on the apical membrane. Fusion of the structure onto CD4 was sufficient to change a random membrane trafficking and expression pattern into a basolateral membrane one. Although the sorting domain contains a PDZ binding motif, this signal is not sufficient to direct basolateral targeting; channels harbouring an internal deletion that leaves the PDZ binding domain intact are still mistargeted to the apical membrane. Nevertheless, the PDZ binding domain plays an important role in basolateral membrane expression. Channels missing the small PDZ ligand accumulate into endosomes, suggesting the PDZ interactions might stabilize basolateral expression by limiting endosomal retrieval. Using the yeast two-hybrid system, we identified a new human orthologue of a C. elegans PDZ protein, hLin7-b, which interacts with the C-terminal tail of Kir2.3. The Lin7 protein co-immunoprecipitates with Kir2.3 and co-localizes with the channel in the cortical collecting duct as a part of a multimeric complex on the basolateral membrane similar to a complex in C. elegans, indicative of an evolutionarily conserved retention machine. The discovery identifies one component of the sorting machinery and provides compelling evidence for a retention mechanism in a hierarchical basolateral trafficking program.

The apical membrane K_ATP channel interacts with other PDZ proteins to co-ordinate subunit assembly. Like other K_ATP channels, the apical channel comprises a Kir tetramer modified by ATP binding cassette subunits. Functional reconstitution studies, supported by co-immunolocalization, suggest that the native channel may be composed of ROMK (Kir1.1) and CFTR. The C-terminal regions of ROMK and CFTR have different binding motives for PDZ interaction, providing a potential scaffolding mechanism for subunit assembly. Using a GST pull-down strategy, NHERF1 and NHERF2 were identified as potential PDZ-dependent binding partners of ROMK in the kidney. Although both co-immunoprecipitate with ROMK in heterologous expression systems, only NHERF2 co-localizes with ROMK in the collecting duct, suggesting that NHERF2 is the physiological binding partner. NHERF contains two PDZ domains. In vitro protein interaction and yeast two-hybrid studies revealed that CFTR preferentially binds to PDZ1 while ROMK specifically binds to PDZ2, indicating that the NHERF has the capacity to simultaneously bind ROMK and CFTR. Indeed, co-expression of NHERF dramatically increased the physical and functional interaction of CFTR with ROMK, as evidenced by an increase in the amount of ROMK that co-precipitates with CFTR and the number of ROMK channels that acquire sensitivity to sulfonylurea agents.

In summary, we have found that PDZ proteins interact with the cortical collecting duct potassium channels to control polarized targeting, retention and subunit assembly. We speculate that these PDZ proteins provide focal points for the formation of signalling complexes that regulate channel number and activity at polarized membrane domains.